The present invention relates to a gel electrophoresis system for separating and analyzing a biological sample.
Electrophoresis is a method for separating samples, by utilizing the difference in the mobility of the samples in a separation medium, depending on the nucleotide sequence or nucleotide length of a nucleic acid sample or the charge or molecular weight of a biological protein sample. The method is useful as a separation technique in the field of biochemistry. Among them, some of nucleic acid separation is for the purpose of electrophoresis for DNA sequencing or for the purpose of identification of an amplified product after PCR reaction. In this case, the electrophoresis frequently uses slab gel. Essentially, the system of slab gel electrophoresis comprises a gel for separating a sample, glass plates for supporting the gel from both sides thereof, and a pair of electrodes and a power source for applying an electric field through a buffer solution to the gel.
So as to carry out electrophoretic analysis with high resolution at high reproducibility, electrophoretic conditions such as applied voltage and gel composition should be maintained constant, but the gel temperature during electrophoresis is one of difficult-to-control conditions with serious effects on sample separation. The temperature control then has two significant meanings; the gel temperature should be retained uniformly and the gel should be retained at an appropriate temperature.
As to the former one, firstly, non-uniform temperature distribution readily develops in the gel, and therefore, a phenomenon has been remarked problematically such that even the same sample shows a different profile of mobility in some case. The phenomenon is generally called as "smiling", and this problem of non-uniform temperature distribution in the gel occurs because the Joule's heat generated via the passing of elecric current in the gel is not uniformly radiated. It is generally supposed that the reason why the temperature then affects the sample mobility resides in that the viscosity of the buffer solution in the gel varies depending on the temperature.
The latter necessity to control the gel temperature at an appropriate temperature is significant when the electrophoretic temperature for optimum separation varies depending on the sample or the separation method. For example, electrophoresis should be carried out satisfactorily at a temperature of 45 to 55.degree. C. for DNA sequencing; for the SSCP method described in Genomics, Vol. 5, pp. 874-879, 1989, alternatively, the temperature should be retained generally at a predetermined temperature from 4 to 40.degree. C.
Because the SSCP method is a separation method based on the phenomenon that a single-stranded DNA forms a conformation depending on the nucleotide sequence and the mobility during electrophoresis varies depending on the resulting conformation, the method requires that the hydrogen bonding between the nucleotides to maintain the conformation functions in a stable manner. On the contrary, separation methods without utilizing such conformation require a temperature at which not any such conformation is formed.
The SSCP method will now be described in detail below. The optimum temperature for good separation varies depending on the nucleotide length and nucleotide sequence of a sample to be separated, and therefore, the temperature for electrophoresis largely influences the separation outcome. Hence, it is very important to control appropriately and strictly the temperature during electrophoresis.
Another reason requiring the temperature control lies in the demand toward high-speed separation. Following the recent technological progress, the increase of the number of genes to be analyzed demands to shorten the analysis time, which is very readily attained by the high applied voltage for electrophoresis. However, such high-voltage application increases the generation of Joule's heat, thereby raising the gel temperature, which means that more efficient cooling and more precise temperature control will be inevitable than has been attained conventionally.
As has been described above, temperature control of the gel during electrophoresis is a very significant issue from the respect of the demands toward separation at high resolution and high speed. So as to overcome the problem, a means of cooling a slab gel during electrophoresis should be arranged additionally on the essential structure of the system. As disclosed in Japanese Patent Laid-open Nos. Sho 57-163861 and 61-57848, conventionally, a number of methods by means of thermostat plate have been invented.